Gel purification of gDNA for next-generation sequencing applications.

We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This method yields a substantial amount at microgram levels of gDNA per gel cleanup with high purity. An RNase A treatment step can be omitted. The quality of gDNA is suitable for next-generation sequencing, resulting in >10 Mb reads and high-quality read data (Phred score >28 up to 100 of 150 base reads). Furthermore, the gDNA can be kept intact in a gel slice for several days. This method has been tested for dictyostelids, bacteria and plants.

Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media.

Background and Objectives: House-keeping genes are generally selected as reference genes in gene expression analysis. However, some genes may not be stably expressed across all experimental conditions. Thus, this study aimed to validate seven house-keeping genes for gene expression analysis in Bacillus siamensis 1021. Materials and Methods: Strain 1021 was grown in potato dextrose broth, nutrient broth and mineral salt medium. Reverse-transcription quantitative PCR was used to determine Cq values of seven reference genes including gyrA, gyrB, ssb and dnaB, rpsU, gat_Yqey and udp in these media. Expression stability of these genes was analyzed, using geNorm and Normfinder applications. The target gene ftsZ was used for assessment of the best candidate genes. Results: Based on geNorm and Normfinder, ssb was the most-stably expressed gene, while udp was the least-stably expressed gene. Pairwise variation indicated the combination of ssb, gyrA, gyrB and gatB_Yqey was suitable for the normalization of ftsZ expression. ftsZ expression in potato dextrose broth and mineral salt medium was higher than that in nutrient broth. In contrast, the normalization against udp resulted in an under- and overestimation of ftsZ expression in potato dextrose broth and mineral salt medium, respectively. Conclusion: The combination of ssb, gyrA, gyrB and gatB_Yqey was the best candidate for normalization of target gene expression in B. siamensis 1021 in these media. This study emphasized the significance of reference gene validation for gene expression analysis and provided a guideline for future gene expression studies in B. siamensis.

Chitinophaga oryzae sp. nov., an epiphytic bacterium isolated from rice root surfaces.

Two Gram-stain-negative, non-motile, rod-shaped bacterial strains were isolated from the surfaces of rice roots. They were designated as strains 1303T and 1310. Their colonies were circular, entire, opaque, convex and yellow. They were chitinase- and catalase-positive, reduced nitrate and grew at 16-37 °C (optimum, 30 °C), pH 5.0-10.0 (optimum, pH 7.0) and 0-2.0% NaCl (optimum, 1.0 %). Based on the 16S rRNA gene sequence analysis, they were classified as members of the genus Chitinophaga. Results of phylogenetic and phylogenomic analyses indicated that they formed a cluster with Chitinophaga eiseniae YC6729T, Chitinophaga qingshengii JN246T, Chitinophaga varians 10-7 W-9003T and Chitinophaga fulva G-6-1-13T. When the genomic sequences of strains 1303T and 1310 were compared with their close relatives, the average nucleotide identity and digital DNA-DNA hybridization values were below the cut-off levels. Phosphatidylethanolamine was the major polar lipid. MK-7 was the major respiratory quinone. iso-C15 : 0, C16 : 1 ω5c, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c) were the predominant fatty acids. Differential characteristics between both strains and their close relatives were also observed. Based on the distinctions in genotypic, phenotypic and chemotypic features, strains 1303T and 1310 represent members of a novel species of the genus Chitinophaga, for which the name Chitinophaga oryzae sp. nov. is proposed. The type strain is 1303T (=KACC 22075T=TBRC 12926T).

Differential effects of synthetic media on long-term growth, starch accumulation and transcription of ADP-glucosepyrophosphorylase subunit genes in Landoltia punctata.

Murashige & Skoog (MS) and Hoagland's media were previously used for in vitro culture of Landoltia punctata. During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland's medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland's medium. Conversely, starch content in MS medium was considerably higher than in Hoagland's medium. Medium effects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Differential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefficients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland's medium, the coefficients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (-0.62). This suggested different levels of contributions of these genes in starch biosynthesis in both media.

Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

Pseudoxanthomonas helianthi sp. nov., isolated from roots of Jerusalem artichoke (Helianthus tuberosus).

A bacterium designated as strain roo10T was isolated from roots of Jerusalem artichoke (Helianthus tuberosus). Cells were Gram-stain-negative and non-motile rods. The phylogenetic analysis of the 16S rRNA gene indicated that it represented a member of the genus Pseudoxanthomonas, and its close relatives included Pseudoxanthomonas kalamensis JA40T (97.8 % 16S rRNA gene sequence similarity), Pseudoxanthomonas sangjuensis 5GH38-5T (97.7 %) and Pseudoxanthomonas daejeonensis TR6-08T (97.1 %). Growth of roo10T occurred at pH 7-9. The temperature for growth ranged from 20 to 37 °C. Tolerance to NaCl was observed from 0.005 to 5 % (w/v) concentration. Predominant fatty acids were iso-C15 : 0 (23.5 %), iso-C16 : 0 (18.9 %) and anteiso-C15 : 0 (11.5 %). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidyl-N-methylethanolamine were the major polar lipids. The predominant quinone was ubiquinone 8 (Q-8). The DNA G+C content was 65.7 mol% [from melting temperature (Tm)]. Comparison of phenotypic and chemotaxonomic characteristics indicated that roo10T was distinguishable from its close relatives. Additionally, the DNA-DNA relatedness levels between roo10T and P. kalamensis DSM 18571T (22±0.5 %), P. sangjuensis 5GH38-5T (21±0.2 %) and P. daejeonensis DSM 17801T (3±1 %) were lower than 70 %. These results indicated that roo10T represented a novel species of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas helianthi sp. nov. is proposed. The type strain is roo10T (=BCC 70700T=NBRC 110414T).

Antimicrobial compounds from endophytic Streptomyces sp. BCC72023 isolated from rice (Oryza sativa L.).

An endophytic actinomycete strain BCC72023 was isolated from rice (Oryza sativa L.) and identified as the genus Streptomyces, based on phenotypic, chemotaxonomic and 16S rRNA gene sequence analyses. The strain showed 99.80% similarity compared with Streptomyces samsunensis M1463(T). Chemical investigation led to the isolation of three macrolides, efomycins M (1), G (2) and oxohygrolidin (3), along with two polyethers, abierixin (4) and 29-O-methylabierixin (5). To our knowledge, this is the first report of efomycin M being isolated from a natural source. The compounds were identified using spectroscopic techniques and comparison with previously published data. All compounds exhibited antimalarial activity against the Plasmodium falciparum, K-1 strain, a multidrug-resistant strain, with IC50 values in a range of 1.40-5.23 µg/ml. In addition, these compounds were evaluated for biological activity against Mycobacterium tuberculosis, Bacillus cereus, Colletotrichum gloeosporioides and Colletotrichum capsici, as well as cytotoxicity against both cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells.

Micromonospora soli sp. nov., isolated from rice rhizosphere soil.

An actinomycete strain SL3-70(T) was isolated from a rice field and characterised using a polyphasic approach. The morphological and chemotaxonomical characteristics of strain SL3-70(T) indicate that it belongs to the genus Micromonospora. The phylogenetic analysis of the nearly complete 16S rRNA gene sequence revealed that strain SL3-70(T) is a member of the genus Micromonospora, and is closely related to Micromonospora echinaurantica DSM 43904(T) (99.1 % 16S rRNA gene sequence similarity) and Micromonospora kangleipakensis MBRL 34(T) (98.8 %). DNA-DNA relatedness between strain SL3-70(T) and its relatives ranged from 21.2 % ± 0.6 to 38.7 % ± 0.4. The results obtained from our study indicate that strain SL3-70(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora soli sp. nov. is proposed. The type strain is SL3-70(T) (=BCC 67268(T); =NBRC 110009(T)).

Micromonospora oryzae sp. nov., isolated from roots of upland rice.

An actinomycete strain, designated CP2R9-1T, was isolated from root internal tissues of upland rice (Oryza sativa). Based on a polyphasic approach, strain CP2R9-1T was characterized as a member of the genus Micromonospora. meso-Diaminopimelic acid and 3-OH-diaminopimelic acid were present in the cell-wall peptidoglycan. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, two unidentified phospholipids and four unidentified polar lipids. Predominant menaquinones were MK-9(H4), MK-9(H6) and MK-10(H4). Whole-cell sugars consisted of ribose, xylose, arabinose and glucose. Phylogenetic analysis of the nearly complete 16S rRNA gene sequence suggested that strain CP2R9-1T was closely related to Micromonospora haikouensis 232617T (99.32 % similarity), Micromonospora carbonacea DSM 43168T (99.18 %) and Micromonospora krabiensis MA-2T (99.16 %). Strain CP2R9-1T was distinct from its closest relatives based on low levels of DNA-DNA relatedness (21.3 ± 0.1-41.7 ± 0.7 %) and phenotypic differences. The results presented in this study showed that strain CP2R9-1T represents a novel species of the genus Micromonospora, for which the name Micromonospora oryzae sp. nov. is proposed. The type strain is CP2R9-1T ( = BCC 67266T = NBRC 110007T).

Micromonospora endophytica sp. nov., an endophytic actinobacteria of Thai upland rice (Oryza sativa).

An actinobacterial strain, DCWR9-8-2(T), was isolated from a leaf of Thai upland rice (Oryza sativa) collected in Chumporn province, Thailand. Strain DCWR9-8-2(T) is Gram-stain-positive aerobic bacteria that produce single spores directly on the vegetative hypha. Cell wall peptidoglycan of this strain exhibits meso-diaminopimelic acid and glycine, the reducing sugars of whole-cell hydrolysate are arabinose, glucose, ribose, xylose and small amount of mannose. The phospholipid profiles in the membrane are comprised of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides. The major menaquinones are MK-9(H4) and MK-10(H6). The diagnostic cellular fatty acids are iso-C16:0 and iso-C15:0. The G+C content of the genomic DNA is 72.5 mol%. The result of 16S rRNA sequence analysis of the strain revealed that this strain was closely related to Micromonospora auratinigra TT1-11(T) (99.25%). On the other hand, the result of gyrB gene sequence analysis revealed that this strain was closed to M. eburnea JCM 12345(T) (96.30%). In addition, a combination of DNA-DNA hybridization results and some phenotypic properties supported that this strain should be judged as a novel species of the genus Micromonospora, for which the name M. endophytica sp. nov. is proposed. The type strain is DCWR9-8-2(T) (=BCC 67267(T)=NBRC 110008(T)).

Paenibacillus lemnae sp. nov., an endophytic bacterium of duckweed (Lemna aequinoctialis).

A Gram-stain-variable, rod-shaped and endospore-forming bacterium, designated strain L7-75, was isolated from duckweed (Lemna aequinoctialis). Cells were motile with a monopolar flagellum. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L7-75(T) belonged to the genus Paenibacillus, and the closest phylogenetically related species were Paenibacillus uliginis N3/975(T) (98.5% 16S rRNA gene sequence similarity), Paenibacillus purispatii ES_M17(T) (98.5%), Paenibacillus lactis MB 1871(T) (98.2%), Paenibacillus campinasensis 324(T) (97.7%), Paenibacillus glucanolyticus S93(T) (97.7%) and Paenibacillus lautus ATCC 43898(T) (97.4%). Growth of strain L7-75(T) was observed at pH 7-10 and at 20-40 °C, and NaCl concentrations up to 5% (w/v) were tolerated. Major cellular fatty acids included anteiso-C15 : 0, C16 : 0 and anteiso-C17:0 that were present at 36.0%, 14.2 % and 10.0% of the total cellular fatty acid profile, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidyl-N-methylethanolamine. MK-7 was the predominant menaquinone. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The DNA G+C content was 49.1 mol% (Tm). DNA-DNA relatedness values between strain L7-75(T) and its closest relatives ranged from 4.4 to 47.8%. These results indicate that strain L7-75(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus lemnae sp. nov. is proposed. The type strain is L7-75(T) ( = BCC 67838(T) = NBRC 109972(T)).

Rhizobium lemnae sp. nov., a bacterial endophyte of Lemna aequinoctialis.

Bacterial strain L6-16(T) was isolated from Lemna aequinoctialis. Cells were Gram-stain-negative, rod-shaped and motile with monopolar flagella. The phylogenetic analysis of its nearly complete 16S rRNA gene sequence revealed that strain L6-16(T) was a member of the genus Rhizobium. Its closest relative was Rhizobium tarimense PL-41(T) with a 16S rRNA gene sequence similarity value of 98.3%. Sequence similarity analysis of the housekeeping recA and atpD genes showed low levels of sequence similarity (<93.9%) between strain L6-16(T) and other species of the genus Rhizobium. Strain L6-16(T) was able to grow between pH 5 and 11 (optimum 7.0) and at temperatures ranging from 20 to 41 °C (optimum 30 °C). It tolerated NaCl up to 1 % (w/v) (optimum 0.5%). C18 : 1ω7c and/or C18 :  1ω6c (summed feature 8; 79.5%) were found as predominant cellular fatty acids. The DNA G+C content of strain L6-16(T) was 58.1 mol% (Tm). Based on low levels of DNA-DNA relatedness, strain L6-16(T) was distinct from members of phylogenetically related species including R. tarimense PL-41(T) (38.3 ± 0.8%), Rhizobium rosettiformans W3(T) (6.9 ± 0.4%) and Rhizobium pseudoryzae J3-A127(T) (12.3 ± 0.6 %). Strain L6-16(T) was unable to nodulate the roots of Phaseolus vulgaris, and nodC and nifH genes were not detected. The results obtained from phylogenetic analyses, phenotypic characterization and DNA-DNA hybridization indicated that strain L6-16(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium lemnae sp. nov. is proposed. The type strain is L6-16(T) ( = NBRC 109339(T) = BCC 55143(T)).

Evaluations of the mutagenicity of a pigment extract from bulb culture of Hippeastrum reticulatum.

The use of anthocyanins in food products as colorants has been limited because of their instability toward alkaline pH and high temperature. This study aimed to determine color stability and mutagenicity of the anthocyanin-based pigment extract from bulb cultures of Hippeastrum (Hippeastrum reticulatum). The pigment extract retained its reddish-orange color under alkaline conditions (⩽pH 11) and was stable up to 6 h at 95 °C. The mutagenicity of the extract was evaluated in vitro and in vivo. Hippeastrum pigment extract up to 1.25 mg plate(-1) was found non-mutagenic in Ames test using Salmonella typhimurium strain TA98 and TA100. Chromosome aberrations were observed when human lymphocytes were treated with the extract up to 1.5 mg ml(-1). However, the extract up to 1.4 mg ml(-1) was found to exhibit relatively low or no mutagenicity in in vitro comet assays with human lymphocytes. In in vivo micronucleated reticulocyte assay, mice were treated orally with the extract up to 1 g kg(-1). No significant increase of the percentage of micronucleated peripheral reticulocytes compared to the negative control groups was found. Taken together, our study indicates that Hippeastrum pigment extract is potentially applicable as an additive colorant in the diet and related products.

Rhizobium paknamense sp. nov., isolated from lesser duckweeds (Lemna aequinoctialis).

A Gram-stain-negative, rod-shaped bacterium was isolated and designated strain L6-8(T) during a study of endophytic bacterial communities in lesser duckweed (Lemna aequinoctialis). Cells of strain L6-8(T) were motile with peritrichous flagella. The analysis of the nearly complete 16S rRNA gene sequence indicated that strain L6-8(T) was phylogenetically related to species of the genus Rhizobium. Its closest relatives were Rhizobium borbori DN316(T) (97.6 %), Rhizobium oryzae Alt 505(T) (97.3 %) and Rhizobium pseudoryzae J3-A127(T) (97.0 %). The sequence similarity analysis of housekeeping genes recA, glnII, atpD and gyrB showed low levels of sequence similarity (<91.5 %) between strain L6-8(T) and other species of the genus Rhizobium with validly published names. The pH range for growth was 4.0-9.0 (optimum 6.0-7.0), and the temperature range for growth was 20-45 °C (optimum 30 °C). Strain L6-8(T) tolerated NaCl up to 2 % (w/v) (optimum 1 % NaCl). The predominant components of cellular fatty acids were C19 : 0 cyclo ω8c (31.32 %), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 25.39 %) and C16 : 0 (12.03 %). The DNA G+C content of strain L6-8(T) was 60.4 mol% (Tm). nodC and nifH were not amplified in strain L6-8(T). DNA-DNA relatedness between strain L6-8(T) and R. borbori DN316(T), R. oryzae Alt505(T) and R. pseudoryzae J3-A127(T) was between 11.2 and 18.3 %. Based on the sequence similarity analyses, phenotypic, biochemical and physiological characteristics and DNA-DNA hybridization, strain L6-8(T) could be readily distinguished from its closest relatives and represents a novel species of the genus Rhizobium, for which the name Rhizobium paknamense sp. nov. is proposed. The type strain is L6-8(T) ( = NBRC 109338(T) = BCC 55142(T)).

Plastid marker gene excision by the phiC31 phage site-specific recombinase.

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.